GSM5359799: Pol II ChIP from Mod(mdg4)-N-KD rep1; Drosophila melanogaster; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
mod(mdg4)
Cell type
Cell type Class
Cell line
Cell type
OSC
Tissue Source
ovary
Developmental Stage
adult stage
Attributes by original data submitter
Sample
source_name
Mod(mdg4)-N-KD OSC
cell type
Ovarian Somatic Cells (OSC)
treatment
RNAi Mod(mdg4)-N
tissue
n/a
genotype
n/a
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP experiments of Ty1-tagged Mod(mdg4) isoforms are performed by truChIP Chromatin Shearing Kit according to manufactures instruction with minor modifications. 2x107 OSC were fixed with 1% formaldehyde 10min and quenched and lysed with buffers of truChIP Chromatin Shearing Kit. Fixed chromatin is sheered with Bioruptor II (BMbio BR2012A) for 20 cycles of 30s ON/30s OFF, high settings. Sheered chromatin is immunoprecipitated by antibody-conjugated Dynabeads protein G for 2hr. Immunoprecipitated chromatin was incubated with Proteinase K and RNase, and de-crosslinked at 65°C overnight. For ChIP experiments of RNA polymerase II, 2x107 OSC were fixed with 1% formaldehyde 10min and quenched with final 0.1M Glycine for 5min. After twice wash with PBS, nuclei were isolated with 1ml of swelling buffer (25mM HEPES-KOH(pH 7.5), 1.5mM MgCl2, 10mM KCl, 0.1% NP-40, 1mM DTT and 1x Protease Inhibitor). Isolated nuclei were lysed in 400µl of sonication buffer(50mM HEPES-KOH(pH 7.4), 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS and 1xProtease Inhibitor) and were sheered with Bioruptor II (BMbio BR2012A) for 20 cycles of 30s ON/30s OFF, high settings. 3µg of antibody were incubated with chromatin for overnight. Next day, 20µl of Dynabeads M-280 Sheep anti-Mouse IgG is added to sample and incubated for 1hr. Beads were washed and treated with Proteinase K and RNase as described (Haring et al., 2007). DNA was purified with isopropanol precipitation using Pellet Paint NF Co-precipitant (Merck: 70748). Fragments from the ChIP experiment were sheared to ∼200 bases using Covaris S220. These were used for library preparation with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacturer's protocol.